Age-related changes in protein-protein interactions in the lens play a critical role in the temporal evolution of its optical properties. In the relatively non-regenerating environment of the fiber cells, a critical determinant of these interactions is partial or global unfolding as a consequence of post-translational modifications or chemical damage to… Read More

To further develop the mechanistic understanding of small heat-shock protein (sHSP) chaperone activity, we investigate the nature of the intermediate states recognized by alpha-crystallin and the conformations that are stably bound. The model substrates consist of a set of well characterized, destabilized T4 Lysozyme (T4L) mutants that have been shown… Read More

Site-directed spin labeling (SDSL) was used to explore the structural framework responsible for the obligatory drug-proton exchange in the Escherichia coli multidrug transporter, EmrE. For this purpose, a nitroxide scan was carried out along a stretch of 26 residues that include transmembrane segment 1 (TMS1). This segment has been implicated… Read More

The consequences of alphaB-crystallin phosphorylation on its chaperone activity were investigated using a detailed analysis of the recognition and binding of destabilized T4 lysozyme (T4L) mutants by alphaB-crystallin phosphorylation mimics containing combinations of serine to aspartate substitutions. The T4L site-directed mutants were selected to constitute an energetic ladder of progressively… Read More

To elucidate the mechanism of alphaA-crystallin chaperone function, a detailed thermodynamic analysis of its binding to destabilized, site-directed mutants of T4 lysozyme was carried out. The selected mutants form a ladder of stabilities spanning the 5-10 kcal/mol range of free energy of unfolding. The crystal structures of the majority of… Read More

The determinants of the oligomeric assembly of Hsp16.5, a small heat-shock protein (sHSP) from Methanococcus jannaschii, were explored via site-directed truncation and site-directed spin labeling. For this purpose, subunit contacts around the two-, three- and four-fold symmetry axes were fingerprinted using patterns of proximities between nitroxide spin labels introduced at… Read More

We report the use of a novel pulsed ESR technique for distance measurement, based on the detection of double quantum coherence (DQC), which yields high quality dipolar spectra, to significantly extend the range of measurable distances in proteins using nitroxide spin-labels. Eight T4 lysozyme (T4L) mutants, doubly labeled with methanethiosulfonate… Read More

We present a novel method for mapping proximity within proteins. The method exploits the quenching of the fluorescent label bimane by nearby Trp residues. In studies of T4 lysozyme we show that this effect appears to be distance dependent and orientation specific. Specifically, we show that a proximal Trp residue… Read More

To explore the spatial organization and functional dynamics of the citrate transport protein (CTP), a nitroxide scan was carried out along 22 consecutive residues within the fourth transmembrane domain (TMDIV). This domain has been implicated as being of unique importance to the CTP mechanism due to (i) the presence of… Read More

The small heat-shock proteins (sHSPs) form a diverse family of proteins that are produced in all organisms. They function as chaperone-like proteins in that they bind unfolded polypeptides and prevent uncontrolled protein aggregation. Here, we present parallel cryo-electron microscopy studies of five different sHSP assemblies: Methanococcus jannaschii HSP16.5, human alphaB-crystallin,… Read More